International Journal of Neuropsychopharmacology (2013), 16, 913–918. f CINP 2012 doi:10.1017/S146114571200096X
B R I EF R E P O R T
Region-specific effects on BDNF expression after contingent or non-contingent cocaine i.v. self-administration in rats Fabio Fumagalli1*, Federico Moro2, Lucia Caffino1, Alessandro Orru`2, Chiara Cassina2, Giuseppe Giannotti1, Angelo Di Clemente2, Giorgio Racagni1,3, Marco A. Riva1 and Luigi Cervo2* 1
Centro di Neurofarmacologia, Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita` degli Studi di Milano, Milano, Italy Experimental Psychopharmacology, Department of Neuroscience, ‘ Mario Negri ’ Institute for Pharmacological Research, Milan, Italy 3 IRCCS San Giovanni di Dio-Fatebenefratelli, Brescia, Italy 2
Abstract Brain-derived neurotrophic factor (BDNF) dynamic changes were investigated in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) during use and the early phases of cocaine abstinence after 14 sessions (2 h self-administration/d ; 0.25 mg/0.1 ml.6 s infusion) by employing a ‘ yoked control-operant paradigm’. The effect on BDNF was region-specific and dependent on the withdrawal time. In the NAc, BDNF protein levels increased immediately after the last self-administration session, with a larger increase in passively cocaineexposed rats. In the mPFC, BDNF expression was elevated 24 h after the last self-administration session, independently of how the drug was encountered. No changes were found in NAc and mPFC 7 d after the last self-administration session. Analysis of transcript levels in the mPFC indicated that action on exon I might contribute to BDNF’s cortical induction. These findings indicate a finely tuned modulation of BDNF expression during use and early phases of cocaine abstinence. Received 6 April 2012 ; Reviewed 7 June 2012 ; Revised 2 July 2012 ; Accepted 30 July 2012 ; First published online 20 November 2012 Key words : BDNF, cocaine, medial prefrontal cortex, nucleus accumbens, self-administration.
Introduction An essential step to develop therapeutic approaches for cocaine addiction is to understand the cellular mechanisms underlying the craving and relapse after withdrawal. Growing evidence suggests that cocaine-induced alterations of brain-derived neurotrophic factor (BDNF) mRNA and protein levels in mesocorticolimbic nuclei are vital in cocaine craving and relapse (Ghitza et al. 2010 ; McGinty et al. 2010). In fact, cue-induced cocaine-seeking progressively increased during protracted abstinence, paralleled by up-regulation of BDNF protein levels in the ventral tegmental area (VTA), nucleus accumbens (NAc) and amygdala (Grimm et al. 2003). Moreover, repeated cocaine self-administration increased BDNF protein levels in the medial prefrontal cortex (mPFC) and VTA after 7 d of abstinence (Sadri-Vakili et al. 2010 ; Schmidt et al. 2012). In Address for correspondence : Dr F. Fumagalli, Centro di Neurofarmacologia, Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita` degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy. Tel. : 0039 0250318298 Fax : 0039 0250318278 Email :
[email protected] * In line with the ICMJE guidelines, these authors are acknowledged as being the guarantors, or lead authors, of this paper and take responsibility for the integrity of the work as a whole, from inception to published article.
addition, a single infusion of BDNF into the mPFC or VTA suppressed the reinstatement of drug-seeking by normalizing cocaine-induced alterations in NAc glutamate transmission (Berglind et al. 2007, 2009 ; McGinty et al. 2010), whereas single or repeated infusions into the NAc and VTA promoted or facilitated cocaine-seeking behaviour (Graham et al. 2007 ; Lu et al. 2004). These findings suggest that self-administered cocaine alters BDNF expression primarily as a result of prolonged drug withdrawal (Grimm et al. 2003 ; Sadri-Vakili et al. 2010). However, Graham et al. (2007) showed an immediate, but transient, increase in BDNF levels in the NAc, suggesting that modulation of BDNF expression after drug withdrawal needs further investigation. Thus, the aim of our study was to investigate : (1) whether dynamic changes of BDNF levels also occurred in early abstinence ; (2) whether such changes rely on the contingency of cocaine self-administration. This is important in light of evidence that acute and chronic cocaine use, even when passively administered, alters BDNF expression in the rat brain (Filip et al. 2006 ; Fumagalli et al. 2007 ; Le Foll et al. 2005). To this aim, by employing the ‘yoked controloperant paradigm ’, in which each rat actively taking cocaine is paired with two yoked controls receiving passive infusions of cocaine or saline (Fumagalli et al. 2009b ; Jacobs et al. 2003), we evaluated the dynamic regulation
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of BDNF expression during use and the early phases of cocaine abstinence after repeated sessions of cocaine self-administration, killing the animals immediately, 24 h or 7 d after the last session. Materials and method Animals Animal procedures were conducted in conformity to institutional guidelines and national (D. L. 116) and international policies (EEC Directive 86/609 ; Guide for the Care and Use of Laboratory Animals, USA). Naive male Sprague–Dawley CD1IGS rats (Charles River, Italy), weighing 250–280 g, were housed individually in a specific pathogen-free vivarium at 21¡1 xC and 60 % humidity under a 12 h inverted light/dark cycle (lights on 07 : 30 hours) with food and water freely available. They were allowed to adapt to these conditions for 2 wk and handled daily during this period. After this, to prevent excessive weight gain they were fed 20 g/animal.d of rat chow in the evening. Rats on this diet gain weight at 1–3 g/d. Cocaine self-administration Self-administration started 7 d after the jugular catheters implantation (Cervo et al. 2007 ; Di Clemente et al. 2012). Rats were randomly assigned to three groups of triads, cocaine self-administration (SA), yoked cocaine (YC) and yoked saline (YS), to be killed immediately, 24 h or 7 d after the last self-administration session. The 2-h SA sessions/d were conducted 7 d/wk for 14 d during the dark phase of the light/dark cycle. The SA procedure has been described in detail previously (Fumagalli et al. 2009b). Briefly, three littermates were trained at the same time. They were individually placed in operant chambers (MED Associates Ltd, USA) controlled by a computer with MED-software. Sessions started with the introduction of two levers and illumination of the house light. The SA rat of each triad was allowed to self-administer i.v. cocaine (0.25 mg/0.1 ml.6 s infusion ; MacFarlan Smith, UK) on a fixed-ratio-1 schedule. Infusions were made by an infusion pump, through a swivel device mounted above the chamber and connected to the external guide cannulae of the catheter. Each infusion was followed by the house light turn-off, retraction of levers and illumination of the stimulus light above the active lever, all of which reverted after 20 s. Further depression of the active lever triggered the same sequence of events. Pressure on the other, inactive lever had no programmed consequences. In half the chambers, the right-hand lever was active, in the others the left-hand one. Cocaine hydrochloride solution was freshly re-mixed every 4 d, filtered through a 20-mm syringe filter and stored at 4 xC. The other two littermates received a responseindependent infusion either of the same dose of cocaine (YC) or saline (YS) accompanied by the same sequence
of events as above, whenever the SA rat did. At the scheduled times after the last self-administration, rats were killed by decapitation. Their brains were quickly removed and the mPFC (including cingulate, infralimbic, prelimbic cortex subregions) was quickly dissected on ice from a 2 mm coronal section extending from approximately bregma +5.16 to +3.24 (Paxinos & Watson, 2005). The NAc (including NAc shell and NAc core subregions) was dissected from a 2 mm section immediately caudal to the mPFC section. Tissues were immediately frozen on dry ice and stored at x80 xC until assay. Preparation of protein extracts and Western blot analysis The preparation of the homogenate and crude synaptosomal fractions from mPFC and NAc as well as the Western blot procedure was as previously described (Fumagalli et al. 2009a). Total protein content (BDNF precursor and mature forms) was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Italy). Total protein concentrations were adjusted to the same amount for all samples (10 mg/lane). BDNF precursor (proBDNF) and mature BDNF (mBDNF) were detected from the band density at 32 and 14 kDa, respectively, after probing with a polyclonal antibody (1 : 500, overnight, 4 xC ; Santa Cruz Biotechnology, USA). Results were standardized using b-actin as a housekeeping protein, detected by examining the band density at 43 kDa after probing with a monoclonal antibody (1 : 10 000 ; Sigma, Italy). The protein immunocomplexes were visualized by chemiluminescence utilizing the ECL Western Blotting kit (Amersham Life Science, Italy). Analysis of gene expression RNA extraction and real-time polymerase chain reaction were performed as previously described (Fumagalli et al. 2012). Samples were run in 384-well formats in triplicate as multiplexed reactions with a normalizing internal control (36B4). The TaqMan Gene Expression Assays (Applied Biosystem, Italy) used for total BDNF and BDNF transcripts I, IV and VI were as previously described (Fumagalli et al. 2012). Statistical analysis The numbers of active and inactive lever presses were analysed by mixed-factorial analysis of variance (ANOVA) with modality of self-administration (SA, YC, YS) and sessions as between- and within-subject factors, respectively. The amount of cocaine (mg/kg i.v.) selfadministered during the 14 sessions was analysed by mixed-factorial ANOVA using, respectively, the time of death and sessions as between- and within-subjects factors. Molecular data were analysed by one-way ANOVA with modality of cocaine exposure as main factor. When
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Number of sessions Fig. 1. Number of active and inactive lever presses by cocaine self-administration (SA), yoked saline (YS) and yoked cocaine (YC) rats during 14 sessions (a). Amount of cocaine self-administered by rats killed immediately, 24 h or 7 d after the last self-administration session (b). Histograms represent the mean¡S.E.M. of at least 5–7 rats per group. See Material and Method for further details. *p
